CAR-T Cells

Chimeric antigen receptor (CAR) T cells are one of the novel approaches to immunotherapy. The definition of CAR-T cell is modified T cells that can recognize target antigens. In that case, modified T cells can readily and easily recognize target cells no matter where they are in the body and destroy them without harming healthy cells1. With these features, CAR-T cells overshadow conventional approaches to diseases such as cancer, fibrosis, and so on1,2. In this article, the elementary basis of CAR-T cells will be explained briefly and clearly.

A chimeric antigen receptor consists of a target binding domain (single-chain variable fragment ‘scFv’), a spacer domain, a transmembrane domain, a costimulatory domain, and signaling domain. Each domain serves a crucial purpose to get the desired response1,3. The target binding domain, also known as the recognition domain, is derived from monoclonal antibodies; bound via a flexible linker to form scFv4,5. Therefore, CARs are MHC-independent T-cell activators6. The spacer domain, also known as the hinge region, is the unit that connects to the target domain, and transmembrane domain. It is essential for flexibility, CAR expression, signaling, epitope recognition, the strength of activation outputs, and epitope recognition1,3,5. The spacer domain has been considered critical for forming effective immunological synapses. The transmembrane domain enables signal transduction. Moreover, recent studies show that its importance is not only about signaling. The transmembrane domain can regulate CAR-T expression, stability, synapse formation, and CAR-T cell functioning. The costimulatory domain enhances the immune response and the signaling domain activates downstream effects.5,6 

Figure 1: Illustration of chimeric antigen receptor’s domains.

Methodology

The first step of designing CAR-T cells is harvesting the patient’s blood to get enough amount T cells (this step is also called leukapheresis or apheresis).1 The next step is modifying T cell ex vivo conditions. Modification can be performed using viral or non-viral methods.3,6 Viral methods can be expressed as CRISPR/Cas9, transposons, and viruses (especially retroviridae). On the other hand, non-viral methods include in vitro transfer (IVT) mRNA method and transferring of TCR genes via electroporation. Right after transduction, modified T cells will be grown in a medium until they reach an adequate number of CAR-T cells1. So far, it can be understood that the production of CAR-T cells is an autologous process. When all these steps are done, there are 2 steps left. The first step is isolation and sterilization. This step is extremely important for the infusion step. To infuse CAR-T cells into a patient’s bloodstream. The solution of CAR-T cells should be sterile to prevent secondary infections3,4. In addition to these, patients should be taken their ongoing treatments to avoid changes in patients’ prognosis4,7.

Mechanism of Action

CAR-T cells can recognize antigens in an MHC-unrestricted manner. This ability enables CAR-T cells to bind to antigens even during infusion. Docking of scFv to the target antigen causes signaling. As a result of this signal, CAR-T cells become active and proliferate. Thus, the therapy is only given once. Activated CAR-T cell releases chemical agents, for example, granzyme and perforin to kill the target cell3,4,6.

Limitations of CAR T cell Therapy

Limitations of CAR T cell therapy can be divided into 5 different categories in order antigen escape, on-target off-tumor, CAR T cell trafficking, tumor infiltration, immunosuppressive microenvironment, and CAR-T cell-associated toxicities.5  

  1. Antigen Escape: Single-antigen targeted CAR-T cell treatment can lead to downregulation/loss of target antigen.
  2. On-target Off-tumor Effects: The chosen target may be found on different cells. In that case, the cells which have the target antigen on their surfaces are also a target for CAR-T cells.
  3. CAR-T cell Trafficking and Tumor Infiltration: In solid tumors, stroma may make up 90% whereas hematological tumors may have minimal. Tumor stroma possesses the ability to limiting the penetration and mobility of CAR-T cells.
  4. Immunosuppressive Microenvironment: Secretion of immunosuppressive cytokines suppress anti-tumor immunity.
  5. CAR-T cell Associated Toxicities: Increased secretion of cytokine and massive T cell expansion may lead to cytokine release syndrome and neurotoxicity5.

Strategies To Overcome

  1. Antigen Escape: Targeting multiple antigens, dual or tandem CARs.
  2. On-target Off-tumor Effects: Tumor cell antigens should not overcome healthy cell antigens.
  3. CAR-T cell Trafficking and Tumor Infiltration: Local application of therapy may be effective, for instance, intrapleural injection.
  4. Immunosuppressive Microenvironment: Combination immunotherapy with CAR-T cells and checkpoint blockade.
  5. CAR-T cell Associated Toxicities: To ameliorate the effectiveness of the therapy, CAR structure can be altered or can be controlled by suicide genes (CAR “off-switch”)
Figure 2: Demonstration of limitations of CAR-T cell therapy. (Sterner, R. C., & Sterner, R. M. ‘2021’ CAR-T cell therapy: current limitations and potential strategies)

Conclusion

A CAR-T cell is a synthetic receptor and consists of a short-chain variable fragment, spacer domain, transmembrane domain, costimulatory domain, and signaling domain. Every domain provides special and vital property to CARs. Moreover, it provides convenience as it is an autologous treatment. Although the potential of CAR-T cells cannot be ruled out, the safety of therapy due to side effects is controversial. Effective treatment may run some unwanted effects, but new strategies provide a safer pathway. Another challenging issue is antigen selection and affinity to bind. Even though their unique utilization, undesirable effects may occur if CAR-T cells bind more strongly to antigens. If CARs bind weaker, their effectiveness will be low1,3,4. In spite of their low effectiveness on solid tumors, CAR-T cells are remarkable agents for haematological tumors and fibrosis.2,7,8 Despite there are lots of challenges, CAR-T cell engineering keeps moving forward.

References:

  1. Miliotou, A. N., & Papadopoulou, L. C. (2018). CAR T-cell Therapy: A New Era in Cancer Immunotherapy. Current Pharmaceutical Biotechnology, 19(1), 5–18. https://doi.org/10.2174/1389201019666180418095526
  2. Rurik, J. G., Tombácz, I., Yadegari, A., Méndez Fernández, P. O., Shewale, S. V., Li, L., Kimura, T., Soliman, O. Y., Papp, T. E., Tam, Y. K., Mui, B. L., Albelda, S. M., Puré, E., June, C. H., Aghajanian, H., Weissman, D., Parhiz, H., & Epstein, J. A. (2022). CAR T cells produced in vivo to treat cardiac injury. Science, 375(6576), 91–96. https://doi.org/10.1126/science.abm0594
  3. Benmebarek, M. R., Karches, C. H., Cadilha, B. L., Lesch, S., Endres, S., & Kobold, S. (2019). Killing mechanisms of chimeric antigen receptor (CAR) T cells. International Journal of Molecular Sciences, 20(6). https://doi.org/10.3390/ijms20061283
  4. Gök, Ö., & Aslan, A. (2019). Personalized Antigen Receptor with Cell Therapy (CAR-T). Journal of the Institute of Science and Technology, 9(4), 2235–2245. https://doi.org/10.21597/jist.591578
  5. Sterner, R. C., & Sterner, R. M. (2021). CAR-T cell therapy: current limitations and potential strategies. Blood Cancer Journal, 11(4). https://doi.org/10.1038/s41408-021-00459-7
  6. Johnson, P. C., & Abramson, J. S. (2022). Engineered T Cells: CAR T Cell Therapy and Beyond. Current Oncology Reports, 24(1), 23–31. https://doi.org/10.1007/s11912-021-01161-4
  7. Wang, Z., Wu, Z., Liu, Y., & Han, W. (2017). New development in CAR-T cell therapy. Journal of Hematology and Oncology, 10(1), 1–11. https://doi.org/10.1186/s13045-017-0423-1
  8. Shah, N. N., & Fry, T. J. (2019). Mechanisms of resistance to CAR T cell therapy. Nature Reviews Clinical Oncology, 16(6), 372–385. https://doi.org/10.1038/s41571-019-0184-6

Figure References:

  1. https://www.dreamstime.com/car-chimeric-antigen-receptor-t-cell-lymphocyte-have-been-genetically-engineered-to-produce-artificial-use-image234010322, retieved Feb 26, 2022.
  2. Sterner, R. C., & Sterner, R. M. (2021). CAR-T cell therapy: current limitations and potential strategies. Blood Cancer Journal, 11(4). https://doi.org/10.1038/s41408-021-00459-7

Inspector: Furkan EKER

Yorum bırakın

E-posta adresiniz yayınlanmayacak. Gerekli alanlar * ile işaretlenmişlerdir